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Biomedical subjects

Y Katsube

Publications and source records attributed to Y Katsube.

At least 37 records · Page 2Linked to original sources
PubMed1998

Analysis of kappa light chain contribution to anti-DNA antibody activity of a human VH4-21-encoded monoclonal antibody (NE-1) by antibody-phage display technique.

Although contribution of light chain to DNA reactivity of some murine anti-DNA antibodies (Abs) has been demonstrated, similar studies on human anti-DNA Abs are limited. To investigate this contribution, we reproduced Fab molecules on the surface of phages from a human B cell line producing IgM anti-DNA monoclonal Ab (NE-1) by an Ab-phage display technique. Expressed Fab molecules (p4-1 clone) were similar to the parental mAb in their binding activities and idiotypic expression. We constructed a light chain shuffled library containing Vkappa genes derived from peripheral blood lymphocytes of a patient with systemic lupus erythematosus (SLE) in combination with the NE-1 heavy chain gene. After panning to ss- or dsDNA, 7 Fab-phage clones which showed significant bindings to ss- or dsDNA were isolated. Many other Fab-phage clones from the library did not bind to ss- nor dsDNA. Sequence analysis revealed that light chains of the 7 clones are derived from diverse Vkappa germline genes including rarely used ones such as the L5 and A30. Most of the Vk germline genes have been used for previously reported anti-DNA antibodies. These findings suggest that diverse Vkappa genes can pair with the NE-1 heavy chain for anti-DNA Ab activity. In addition, kappa light chains seem to modulate DNA binding activities in various ways.

Amino Acid Sequence↗
PubMed1997

Occurrence of sustained increase in QT dispersion following exercise in patients with residual myocardial ischemia after healing of anterior wall myocardial infarction.

Our objective was to evaluate the effect of exercise on QT dispersion over the next 3 hours, as seen on a standard 12-lead electrocardiogram in patients with healed myocardial infarction with or without residual ischemia. We measured QT and QTc dispersion before, immediately after, and 1 and 2 hours after symptom-limited, dynamic treadmill exercise tests in 28 patients with healed anterior wall myocardial infarction with (group I, n = 18) and without (group II, n = 10) residual ischemia. The same protocol was followed in 5 group I patients after successful performance of coronary angioplasty. QT and QTc dispersion did not change immediately after exercise in group II. These parameters increased in group I (QT dispersion at rest [mean +/- SD] 57 +/- 22 ms, and after exercise 87 +/- 27 ms; QTc dispersion at rest 62 +/- 25 ms, and after exercise 114 +/- 36 ms). The increases in QT and QTc dispersion were sustained for at least 2 hours. After a successful coronary angioplasty in 5 patients, these parameters no longer increased with exercise. Thus, QT dispersion increased for at least 2 hours after exercise in patients who had residual ischemia after healing of myocardial infarction. Data obtained in 5 of these patients after coronary angioplasty support the idea that residual ischemia plays a key role in the sustained increase in QT dispersion after exercise.

Aged↗
PubMed1997

Silent myocardial ischemia in Kawasaki disease: evaluation of percutaneous transluminal coronary angioplasty by dobutamine stress testing.

BACKGROUND: Myocardial ischemia and myocardial infarction are the most serious complications of coronary artery lesions in children with Kawasaki disease (KD). Therefore, early detection and treatment of myocardial ischemia in patients with KD is essential. We studied the effectiveness of percutaneous transluminal coronary angioplasty (PTCA) in patients with silent myocardial ischemia detected by dobutamine stress 99mTc myocardial scintigraphy (TMS), body surface mapping (BMS), and signal-averaged ECG late potentials (ELP). METHODS AND RESULTS: Eight of 76 asymptomatic patients with a coronary stenosis >25% and a positive dobutamine stress test were considered to have silent myocardial ischemia. All eight patients had >95% stenoses demonstrated by coronary angiography (CAG) just before PTCA. After PTCA, CAG showed that all of the coronary artery stenoses had been reduced to <50%. Additionally, intravascular ultrasonography (IVUS) performed in five patients before and after PTCA demonstrated adequate dilation of the coronary stenosis after PTCA. All eight patients underwent dobutamine stress TMS, BMS, and ELP 2 to 3 months after PTCA, which demonstrated no regions of myocardial ischemia. Approximately 6 months later, CAG was performed in all eight patients, and only one patient had developed restenosis. CONCLUSIONS: PTCA effectively dilates stenotic coronary arteries in children with KD. Moreover, dobutamine stress TMS, BMS, and ELP are useful for detecting silent myocardial ischemia and estimating the effectiveness of PTCA. Furthermore, IVUS is useful for evaluating the severity of coronary artery lesions before and after PTCA in patients with KD.

Adolescent↗
PubMed1997

Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose.

The three-dimensional structures of the catalytic residue Glu219-->Gln mutant of Pseudomonas stutzeri maltotetraose-forming exo-alpha-amylase, and its complex with carbohydrate obtained by cocrystallization with maltopentaose were determined. Two crystal forms were obtained for the complexed enzyme, and a bound maltotetraose was found in each. The structures were analyzed at 2.2 A and 1.9 A resolution, respectively for the uncomplexed and complexed mutant. These structures were compared with the wild-type enzyme structure. In the complexed crystals, the maltotetraose was firmly bound, extensively interacting with the amino acid environments in the active cleft. The non-reducing end glucose unit was hydrogen bonded to the side-chain of Asp160 and the main-chain nitrogen of Gly158, which seem to be predominantly required for the recognition of the non-reducing end of the substrate that determines the exo-wise degradation of this enzyme. The reducing end glucose unit of bound maltotetraose showed clear deformation, adopting a half-chair conformation with extensive hydrogen bonds to surrounding polypeptides. The C1-atom of this deformed glucose unit lies very close to Asp193OD1 with a distance of 2.6 A. The catalytic residue Asp294 is firmly hydrogen-bonded to the O2 and O3-hydroxyl groups of the deformed reducing end glucose unit. Upon binding of the carbohydrate, small but significant induced fits were observed in the regions of Asp294, Phe156, Ile157, and Asp160. Possible roles of the three catalytic residues are also discussed.

Amylases↗
PubMed1997

Levosimendan, a novel Ca2+ sensitizer, activates the glibenclamide-sensitive K+ channel in rat arterial myocytes.

The electrophysiological effect of levosimendan, a novel Ca(2+)-sensitizing positive inotropic agent and vasodilator, was examined on rat mesenteric arterial myocytes using the patch clamp technique. Resting potential was significantly hyperpolarized with levosimendan, with an EC50 of 2.9 microM and maximal effect (19.5 +/- 3.5 mV; n = 12) at 10 microM. Levosimendan (10 microM) significantly increased the whole-cell outward current. The currents intersected close to the calculated EK (-84 mV), suggesting that the activated current was a K+ current. Hyperpolarization and stimulation of K+ current by levosimendan were not prevented by 30 microM H-7 (a non-specific inhibitor of protein kinases) and 100 nM charybdotoxin (a blocker of Ca(2+)-activated K+ channels), but were abolished by 10 microM glibenclamide. In single-channel current recording in open cell-attached patches, two types of K+ channels were observed having conductances of 26 and 154 pS. The 154 pS channels were not affected by levosimendan and glibenclamide. The 26 pS channels were evoked in one-fourth of the patches when 10 microM levosimendan (and 0.1 mM UDP) was added (at -60 mV) and channel activity was abolished by glibenclamide. The mean open probability of the 26 pS channels was 0.094 +/- 0.017 (n = 9), and the mean open time (at -60 mV) was 6.6 ms in the presence of UDP and levosimendan. Although significant hyperpolarization (4.7 +/- 1.5 mV, n = 8) was observed at 1 microM levosimendan, the same concentration did not affect Ca2+ channel currents (n = 10). In summary, levosimendan hyperpolarized the arterial myocytes, probably through activation of a glibenclamide-sensitive K+ channel. This mechanism may contribute to the vasodilating action of levosimendan.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization.

The crystal structure of oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refined to 2.0 A resolution with an R-factor of 19.6% for 43,328 reflections. The final model contains 4646 protein atoms and 221 ordered water molecules with respective root-mean-square deviations of 0.015 A for bond lengths and of 3.166 degrees for bond angles from the ideal values. The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558). The N-terminal domain takes a (beta/alpha)8-barrel structure with additions of an alpha-helix (N alpha6') between the sixth strand Nbeta6 and the sixth helix N alpha6, an alpha-helix (N alpha7') between the seventh strand Nbeta7 and the seventh helix N alpha7 and three alpha-helices (N alpha8', N alpha8" and N alpha8'" between the eighth strand Nbeta8 and the eighth helix N alpha8. The subdomain is composed of an alpha-helix, a three-stranded antiparallel beta-sheet, and long intervening loops. The C-terminal domain has a beta-barrel structure of eight antiparallel beta-strands folded in double Greek key motifs, which is distorted in the sixth strand Cbeta6. Three catalytic residues, Asp199, Glu255 and Asp329, are located at the bottom of a deep cleft formed by the subdomain and a cluster of the two additional alpha-helices N alpha8' and N alpha8" in the (beta/alpha)8-barrel. The refined structure reveals the locations of 21 proline-substitution sites that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases with different thermostability. These sites lie in loops, beta-turns and alpha-helices, in order of frequency, except for Cys515 in the fourth beta-strand Cbeta4 of the C-terminal domain. The residues in beta-turns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu487) are all found at their second positions, and those in alpha-helices (Asn109, Glu175, Thr261 and Ile403) are present at their N1 positions of the first helical turns. Those residues in both secondary structures adopt phi and phi values favorable for proline substitution. Residues preceding the 21 sites are mostly conserved upon proline occurrence at these 21 sites in more thermostable Bacillus oligo-1,6-glucosidases. These structural features with respect to the 21 sites indicate that the sites in beta-turns and alpha-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops. This well supports the previous finding that the replacement at the appropriate positions in beta-turns or alpha-helices is the most effective for protein thermostabilization by proline substitution.

Amino Acid Sequence↗
PubMed1997

Crystal structure of a maltotetraose-forming exo-amylase from Pseudomonas stutzeri.

The three-dimensional structure of an exo-type alpha-amylase from Pseudomonas stutzeri which degrades starch from its non-reducing end to produce maltotetraose has been determined by X-ray structure analysis. The catalytic domain of this enzyme (G4-2), whose structure was determined, is a product of spontaneous limited proteolysis in culture broth. It has 429 amino acid residues and a molecular mass of 47,200, and crystallizes in ammonium sulfate solution at pH 7.5. The structure was elucidated by the multiple isomorphous replacement method and refined at 2.0 A resolution, resulting in a final R-factor of 0.178 for significant reflections with a root-mean-square deviation from ideality in bond distances of 0.013 A. The polypeptide chain of this molecule folds into three domains; the first with a (beta/alpha)8 barrel structure, the second with an excursed part from the first one, and the third with five-stranded antiparallel beta-sheets. The active cleft is formed on the C-terminal side of the beta-sheets in the (beta/alpha)8 barrel as in the known endo-type alpha-amylases. A histidine side-chain nitrogen ND1 is coordinated to one of the bound calcium ion. The recognition site of the non-reducing end of the amylose that determines exo-wise degradation is presumed to be at one end of this cleft where there is a disordered loop consisting of the 66th to 72nd residues, and a loop carrying an aspartic acid (Asp160). These structural features may be responsible for the binding of the non-reducing end of the substrate amylose.

Amino Acid Sequence↗
PubMed1997

Disruption of actin cytoskeleton attenuates sulfonylurea inhibition of cardiac ATP-sensitive K+ channels.

Two actin filament-depolymerizing agents, DNase I and cytochalasin D, were used to examine the involvement of the cytoskeleton in the functional interaction between the sulfonylurea receptor (SUR) and the ATP-sensitive K+ (KATP) channels. Isolated rate ventricular cardiomyocytes were studied using open cell-attached patches for single-channel recording. Bath application of DNase I (100 microg/ml) or cytochalasin D (10 microM) stimulated the KATP channel activities (in presence of 30 microM ATP), and these channels became resistant to inhibition by tolbutamide (0.5 mM). After exposure to tolbutamide, the relative NPo value was 0.09 +/- 0.02 in control patches in absence of actin disrupters, and 0.67 +/- 0.22* or 0.65 +/- 0.10*, respectively, in cells treated with DNase I or cytochalasine D (*P < 0.05 vs. control). The inhibitory action of glibenclamide (10 microM) on the KATP channels was also attenuated by DNase I. Thus, the disruption of the actin cytoskeleton attenuates the ability of SUR to inhibit the opening of KATP channels.

Actins↗
PubMed1997

[Distribution of serogroups of Vibrio cholerae non-O1 non-O139 with specific reference to their ability to produce cholera toxin, and addition of novel serogroups].

A total of 1898 strains of Vibrio cholerae non-O1 non-O139, which had been collected worldwide for the past 3 year period of 1994-1996, were serogrouped. The strains were also examined for presence of cholera toxin (CT) gene (ctx) and NAG-ST gene, and strains which carried to ctx were further analyzed for their ability to produce CT. In addition, attempts were made to establish novel serogroups for those serologically untypable strains. Of those examined, 1,774 strains of V. cholerae non-O1 non-O139 was classified into 128 known serogroups while 50 strains were found to belong to R type, and the rest of the 74 strains could not be serotyped. Distribution of the serogroups did not seem to correspond to either the strains geographic distribution or sources of isolation. Of those serologically untypable strains, 38 novel serogroups (O156-O193) were established and added to our reference of V. cholerae antigenic schema. It was also found that antisera raised against many V. cholerae strains included R antibodies. This indicates that any V. cholerae antisera for diagnostic purpose should be absorbed with the reference R strains, CA385, before use. There were luminescence producing strains among those sucrose and VP reaction negative strains. Subsequent DNA/DNA homology analysis revealed that they were identified as V. cholerae. This points to a possibility that strains tentatively identified as Vibrio mimicus by conventional biochemical tests may have included luminescent strains of V. cholerae. It is thus highly recommended that strains in question should be tested for the luminescence production in order to differentiate V. cholerae from V. mimicus. Of those 1989 strains examined, 37 strains (ca. 2%) were found to produce CT. Interestingly, CT producing strains were prevalent in serogroup O141; 10 strains out of 16 strains (63%) were positive for CT. The evidence calls for a caution to possible occurrence of cholera-like diarrhea caused by V. cholerae O141 in the future.

Cholera Toxin↗
PubMed1997

[Studies of Vibrio cholerae O140 (serogroup Hakata) isolated from river water].

Strains of Vibrio cholerae O140, which were isolated from river (Hikichi River) water in Kanagawa, Japan, were studied for their biochemical characteristics and toxin genes, and analyzed by an amplified polymorphic DNA method in comparison with both clinical and environmental isolates of V. cholerae O140. Biochemical characteristics of 4 river isolates studied were found to be unanimous by conventional tests and a commercially available identification kit (Api 50 CH). The strains were also found to react positively with both cholera Inaba factor (C) and V. cholerae O140 specific factor (F). A total of 34 V. cholerae O140 strains including the 4 stains, a reference strains (487-95), 25 strains isolated from samples of river or sea water in Japan, 4 strains isolated from a patient and imported foods, was investigated by the PCR method for presence of ctx, zot, hlyA, and NAG-ST genes. It was found that all strains carried the hlyA gene but none of them carried either ctx, zot, or NAG-ST genes. V. cholerae O140 shared a common antigenic structure with pathogenic V. cholerae strains but its pathogenicity seems to be comparable to that of V. cholerae non-O1 non-O139. Subsequent RAPD analysis indicated that the strains can be divided into 9 groups. Four strains from the Hikichi River showed the same RAPD pattern. The results suggest that the analysis can be a useful epidemiological tool for V. cholerae O140, and that V. cholerae O140 persists in the Hikichi River throughout years.

Japan↗
PubMed1997

Regulation of Ca2+ channel currents by intracellular ATP in smooth muscle cells of rat mesenteric artery.

Regulation of L-type Ca2+ channels of vascular smooth muscle (VSM) cells by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent phosphorylation, which requires Mg2+ATP as a phosphate donor, has been reported (T. Ishikawa, J. R. Hume, and K. D. Keef. Circ. Res. 73: 1128-1137, 1993; Z. Xiong, N. Sperelakis, and C. Fenoglio-Preiser. J. Vasc. Res. 31: 271-279, 1994), and regulation by ATP has been demonstrated (Y. Ohya and N. Sperelakis. Circ. Res. 64: 145-154, 1989). However, it has not been elucidated whether the regulation by ATP is mediated by a mechanism that is distinct from phosphorylation. In the present study, we examined the effects of intracellularly perfused ATP on Ca2+ channel currents of VSM cells isolated from rat mesenteric arteries using a whole cell voltage clamp combined with an intracellular perfusion technique. Ba2+ currents (I(Ba)) through Ca2+ channels were evoked by depolarizing pulses from a holding potential of -80 mV with 130 mM Cs+ in the pipette and 100 mM Ba2+ in the bath. The decrease in the ATP concentration (from 5 to 0.1 mM) in the pipette caused a 45 +/- 5% (n = 8) reduction of maximal I(Ba) obtained at +40 mV within 10 min. The dose-response relation between I(Ba) and ATP showed a dissociation constant of 0.53 mM ATP. This concentration is much higher than that usually required for phosphorylation (e.g., few micromolar). Increase in the ATP (from 0.1 to 5 mM) caused an enhancement of maximal I(Ba) by 57 +/- 10% (n = 6), and this enhancement was not prevented in the presence of 30 microM H-7, a nonspecific inhibitor of protein kinases, or 1 microM protein kinase inhibitor, an inhibitor protein of cAMP-dependent protein kinase. These results indicate that slow Ca2+ channels in VSM cells are regulated by intracellular ATP, independently of phosphorylation, implying a direct regulatory action, such as a requirement for ATP binding to the inner surface of the channel, to exhibit activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

The novel calcium sensitizer levosimendan activates the ATP-sensitive K+ channel in rat ventricular cells.

Levosimendan, a new Ca++-sensitizing and positive inotropic agent, was reported to act as a coronary vasodilator and protect ischemic myocardium. To elucidate the mechanisms of these actions, the possible electrophysiological effects of levosimendan on isolated rat ventricular cells were examined by the patch-clamp technique with whole-cell and single-channel recordings. Levosimendan (3 and 10 microM) markedly shortened action potential duration and activated an outward current at potentials positive to -70 mV. The increased current was abolished by glibenclamide, a blocker of the ATP-sensitive K+ (K[ATP]) current. Stimulation of K[ATP] current was dose dependent, with an EC50 value of 4.7 microM; a maximal effect occurred at 30 microM. The L-type Ca++ current was not affected by levosimendan (0.2-10 microM). In single-channel current recording in open cell-attached patches, K[ATP] channels, which had been inhibited by 0.3 mM ATP, were activated by levosimendan. However, levosimendan did not stimulate the K[ATP] channels that exhibited high spontaneous activity in ATP-free solution. Levosimendan also could not stimulate K[ATP] channels that had rundown in ATP-free solution. However, levosimendan could stimulate rundown K[ATP] channels that were reactivated by nucleotide diphosphates. K[ATP] channels inhibited by 0.5 mM AMP-PNP, a nonhydrolyzable ATP analog, were not stimulated by levosimendan; however, the channels were stimulated by levosimendan in the presence of 30 to 50 microM ADP. Levosimendan stimulates cardiac K[ATP] channels that are suppressed by intracellular ATP. It appears that levosimendan acts synergistically with nucleotide diphosphates. These properties of levosimendan may help protect ischemic myocardium because activation of K[ATP] channels by levosimendan would likely occur in ischemic regions in which intracellular ADP concentration is increased and intracellular ATP concentration is decreased.

Action Potentials↗
PubMed1996

Differences in isoproterenol stimulation of Ca2+ current of rat ventricular myocytes in neonatal compared to adult.

The developmental changes in the isoproterenol stimulation of the L-type calcium current (ICa(L)) were studied in freshly isolated neonatal (3-5-day-old) and adult (2-3-month-old) rat ventricular myocytes using whole-cell voltage clamp (at room temperature). ICa(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300 ms pulse from a holding potential of -40 mV. The pipette solution was Cs(+)-rich and Ca(2+)-free. The external solution was Na(+)-free and K(+)-free. Isoproterenol stimulated ICa(L) in a dose-dependent manner. The concentrations of isoproterenol for half-maximal effect were 6.8 nM in neonatal and 13.3 nM in adult. The maximal stimulation of ICa(L) was 147 +/- 14% in neonatal and 97 +/- 7% in adult. The steady-state inactivation curves were not affected by isoproterenol, whereas the steady-state activation curve was shifted to the left in both neonatal and adult. Forskolin (10 microM) increased ICa(L) by 105 +/- 10% in neonatal and 90 +/- 12% in adult. After stimulating ICa(L) by forskolin, the addition of isoproterenol produced a further increase of ICa(L) by 99 +/- 27% in neonatal, but only by 19 +/- 3% in adult. The presence of an inhibitor of cAMP-dependent protein kinase in the pipette did not affect this marked difference between neonatal (87 +/- 23%) and adult (11 +/- 8%). We conclude that, in rat ventricular myocytes, (1) stimulation of ICa(L) by the beta-adrenoceptor agonist, isoproterenol, is already fully developed in the neonatal stage and actually decreases during development; (2) there is evidence for a cAMP-independent stimulation of Ca2+ channels by isoproterenol, and this is greater in neonatal than in adult. We believe that the cAMP-independent pathway is the direct pathway mediated by Gs alpha protein.

Adrenergic beta-Agonists↗
PubMed1996

Na+/Ca2+ exchange current: lack of effect of taurine.

The intracellular taurine content is very high in some types of cells, including myocardial cells, and taurine becomes depleted in failing hearts. The effect of taurine on inward (forward) and outward (reversal) Na+/Ca2+ exchange current (INa/Ca) was examined in freshly isolated rat (11-20-day-old) ventricular cells using whole-cell voltage clamp. INa/Ca was evoked by a 300 ms ramp hyperpolarizing pulse at a rate of 0.67 V/s (from +60 to -140 mV) from a holding potential of -40 mV. Under the condition of 500 nM Ca2+ in the pipette, when the bath solution was changed from 150 mM Li+ to 150 mM Na+, an inward INa/Ca was initiated. On the other hand, under the condition of 50 mM Na+ and 56 nM Ca2+ in the pipette, when the bath solution was changed from Na(+)-free, Ca(2+)-free solution to Na(+)-free, 2 mM Ca2+, an outward INa/Ca was initiated. Both inward and outward INa/Ca were completely blocked by 5 mM Ni2+, helping to identify the current as INa/Ca. Forward and reversal INa/Ca was measured in the absence (control) and presence of 30 mM taurine added to the bath. The current densities (pA/pF) of inward INa/Ca at -50 mV were 0.75 +/- 0.12 in control and 0.73 +/- 0.11 in presence of 30 mM taurine. Outward INa/Ca at +50 mV were 0.73 +/- 0.08 in control and 0.65 +/- 0.02 in 30 mM taurine. There were no significant differences between control and taurine for both inward and outward INa/Ca. Therefore, the various effects of taurine reported on the heart are not caused by a direct effect of taurine on Na/Ca exchange.

Animals↗
PubMed1996

Decreased baroreflex sensitivity in patients with stable coronary artery disease is correlated with the severity of coronary narrowing.

Although studies have shown that arterial baroreflex sensitivity (BRS) is decreased in patients with acute myocardial infarction, BRS changes in patients with stable coronary artery disease (CAD) have not been studied extensively. We assessed BRS by the phenylephrine method in 55 normotensive and nondiabetic patients with chronic effort angina, old myocardial infarction, or both. The control group consisted of 24 age-matched patients without coronary lesions. To identify factors that determine BRS in stable CAD, we performed multivariate analysis using age, sex, left ventricular ejection fraction, pulmonary artery wedge pressure, resting systolic blood pressure, resting heart rate, the number of stenotic coronary arteries, history of myocardial infarction, and the presence or absence of angina pectoris as variables. BRS was significantly lower in patients with CAD than in control subjects (5.9 +/- 2.9 vs 6.9 +/- 2.4 ms/mm Hg, p < 0.05). In patients with CAD, BRS was inversely correlated with age, the resting heart rate, and the number of stenotic coronary vessels (p < 0.001, p < 0.005, and p < 0.005, respectively), but was independent of other clinical parameters, including the history of myocardial infarction. In control subjects, BRS was significantly correlated only with age. These results indicate that BRS is decreased in patients with stable CAD, and this decrease is correlated with the extent and severity of coronary narrowing.

Age Factors↗
PubMed1996

A pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution.

The crystal structure of glutathione synthetase from Escherichia coli B complexed with ADP, glutathione, and sulfate has been determined at 2.0 A resolution. Concerning the chemical similarity of sulfate and phosphate, this quaternary complex structure represents a pseudo enzyme-substrate complex in the reverse reaction and consequently allows us to understand the active site architecture of the E. coli glutathione synthetase. Two Mg2+ ions are coordinated with oxygen atoms from the alpha- and beta-phosphate groups of ADP and from the sulfate ion. The flexible loops, invisible in the unliganded or the binary and ternary complex structures, are fixed in the quaternary complex. The larger flexible loop (Ile226-Arg241) includes one turn of a 310-helix that comprises the binding site of the glycine moiety of GSH. The small loop (Gly164-Gly167) is involved in nucleotide binding and acts as a phosphate gripper. The side chains of Arg210 and Arg225 interact with the sulfate ion and the beta-phosphate moiety of ADP. Arg 210 is likely to interact with the carboxylate of the C-terminal gamma-glutamylcysteine in the substrate-binding form of the forward reaction. Other positively charged residues in the active site (Lys125 and Lys160) are involved in nucleotide binding, directing the phosphate groups to the right position for catalysis. Functional aspects of the active site architecture in the substrate-binding form are discussed.

Adenosine Diphosphate↗
PubMed1996

Crystal structure of an elastase-specific inhibitor elafin complexed with porcine pancreatic elastase determined at 1.9 A resolution.

The crystal structure of a stoichiometric complex between an elastase-specific inhibitor elafin and porcine pancreatic elastase (PPE) has been determined and refined to a crystallographic R-factor of 19.7% at 1.9 A resolution. The polypeptide chain of elafin has a planar spiral shape with an exposed external part and an internal core part which resembles both the crystal structure of human seminal plasma inhibitor (HUSI-1) [Grütter, M. G., Fendrich, G., Huber, R., & Bode, W. (1988) EMBO J. 7, 345-351] and the solution structure of Na+,K(+)-ATPase inhibitor (SPAI-1) revealed by NMR analysis [Kozaki, T., Kawakami, Y., Tachibana, S., Hatanaka, H., & Inagaki, F. (1994) Pept. Chem., 405-408]. The external region containing the primary binding loop is interconnected by four disulfide bonds to the internal part composed of a beta-sheet and a hairpin loop. The scissile peptide bond Ala24i(P1)-Met25i(P1') in the primary binding site is intact, and its carbonyl carbon is in van der Waals contact with O gamma of the active site Ser195 of PPE. The seven residues of Leu20i(P5)-Leu26i(P2') of the primary binding loop and the three residues of Ser48i, Cys49i, and Ala52i of the adjacent hairpin loop are in contact with PPE by hydrogen bonds and/or van der Waals interactions in a manner similar to that observed for other serine protease-inhibitor complexes. Electron densities of the N-terminal residues Ala1i-Ser10i which are not responsible for the elastase inhibitory activity were not visible, probably due to disordered conformation. The guanido group (N eta 1, N eta 2) of Arg61 in the complex interacts with S delta of Met25i(P1') by possible hydrogen bonds between N and S atoms, accompanying a large positional shift of the side chain of Arg61-(S1') between the complexed and free forms of PPE. The primary binding site is stabilized by hydrogen bonds between the guanido group (N eta 1, N eta 2) of Arg22i(P3) and the carbonyl group of Met25i(P1') across the scissile bond, as well as by a hydrogen bond between the amino group of Cys23i(P2) and the carbonyl group of Ser48i in the internal core. This intramolecular hydrogen bond network and the network of four disulfide bonds might play a significant role in stabilizing the conformation of the binding site for expressing the potent specific inhibitory activity.

Amino Acid Sequence↗